NO120203B - - Google Patents

Description

Process for the production of griseofulvin.

The present invention relates to a method for producing griseofulvin by cultivating organisms under submerged conditions.

Griseofulvin is a well-known antibiotic

means that have been shown to have significant

properties as an agent against fungi. Its structure was first determined by Grove et al. (Chem. & Ind. 17 March 1951, page 219) and the following constitutional formula was given to the compound by the aforementioned researchers:

Production and isolation of griseofulvin on a large scale has so far been difficult. Thus it appears from the literature (see Oxford et al. J. Biochem. XXXIII (2), 240, (1939); Brian et al. Trans. Brit. Mycol. Soc. 29, 173

(1946); Grove and staff Nature 160,

574 (1947); Same Grove and co-workers together with MacMillan, Chem. & Ind. 25 August 1951, (page 719) that griseofulvin has been produced by surface culture only from 3 species of moulds, namely P. griseofulvin, P. janczewskii and P. patulum.

The antibiotic griseofulvin

has, as mentioned, valuable fungicidal properties and is, among other things, very well suited for use as an agent against fungi, especially in agriculture, e.g. in spray liquids for fruits and other crops or as a protective agent for seeds. Practical use of antibiotic agents especially on a large scale such as

in agriculture, requires that this can be produced cheaply in large quantities. The methods proposed so far for the production of griseofulvin all include surface culture of various molds, a technique which on

neither method is suitable for large-scale production.

It has now been found that it is possible to produce griseofulvin on a large scale and in a more convenient way than was previously possible

by growing suitable organisms under

submerge aerobic conditions and by taking careful account of various factors determining the culture.

Different species of the family Penicillium are known to produce griseofulvin in different amounts when grown in

appropriate media. It is difficult to state

exactly which species are suitable for the production of griseofulvin by cultivation under submerged conditions and different strains

more of a particular species varies considerably. The simplest method of determining which strains can be used for a satisfactory culture under submerged conditions is possibly to carry out test cultures in accordance with the method according to the present invention and to select those strains which produce the antibiotic agent in reasonable quantities. The antibiotic agent is mainly found in the mycelium, but the determination of the amount produced is based on the amount of antibiotic agent contained in the entire culture medium. In general, a griseofulvin-producing organism should be used for cultivation which, under deep culture conditions, produces at least 150 micrograms of griseofulvin per ml culture medium. It is hereby assumed that the cultivation is carried out under the conditions stated below and for such a long time until the breakdown of the mycelium just begins. It is desirable to use a strain that produces at least 200 micrograms per ml and it is preferable to use a strain that produces at least 500 micrograms per ml.

A strain which has been found to be particularly suitable for use in the method according to the invention is the strain known as Penicillium patulum Bainier Thorn (4640, 455) C.M.I. 39, 809 [NRRL

(989) as P. urticae Bain (A) — G. Smith 1949. This strain and mutants thereof are currently preferred, as it has been found to give a high yield of griseofulvin under various conditions of submersible cultivation. Furthermore, this strain and mutants thereof have shown no significant tendency to produce dechlor-griseofulvin when grown under the conditions set forth below, even when using assays sensitive to one microgram per ml. As you know, the dechlor compound has a noticeably lower activity against fungi than griseofulvin itself, and is of little value. Other strains that have been tried and found to be more or less suitable are as follows: Asymmetrica Fasciculata ( P. urticae series) :- Penicillium patulum CMI 28, 808 Bainier Coll-Thom (4640-454). NCTC (1722) 1932 — NRRL 994, ATCC 9260

Penicillium urticae Bain, (Rg 8g)

( P. griseofulvum) C.B.S. Baarn Asymmetrica- Divaricata ( P. nigricans series) :-

Penicillium nigricans (Bain) (Zaleski).

CBS Baarn

Penicillium janczew' skii Zal.

C.M.I. 29, 100. (Soil, Lakenheath War-ren,

Suffolk 1947 — J. H. Warcup B25) Penicillium albidum Fungi. C.M.I. 40, 219

Penicillium raciborskii Zal. C.M.I. 40, 568 (?T)

Penicillium melinii Thorn. C.M.I. 40, 216

It has been found that in order to achieve the relatively high yields of griseofulvin which are possible by cultivation under deep culture conditions according to the present invention, the cultivation should take place under certain conditions which are either essential or preferred.

The media used in the method according to the invention should contain a source of nitrogen, a source of carbon and energy and nutritious salts. You can use completely synthetic media (in which, for example, the nitrogen is present as sodium nitrate), but it is preferable to use media in which the nitrogen is obtained from complex organic substances, as such substances often have the advantage that they add growth factors which are often beneficial with regard to supporting good growth of the micro-organisms. Among suitable complex nitrogen-containing materials are e.g. corn extracts "Corn steep liquor (SCL), milk products such as whey powder, buttermilk, liquid whey, cottonseed meal, oat flour and soybean meal, etc. Which of these substances you choose will largely depend on their availability and price, but is also determined by other factors in the cultivation technique used, e.g. the ease with which the pH can be controlled. The final choice should depend on the balance of benefits in the circumstances. You can also achieve good results by using 2 or more different nitrogen sources together, such as e.g. a mixture of grain extracts and whey powder or liquid whey.

As a source of carbon and energy, a carbohydrate such as lactose, glucose, sucrose or starch is preferred. For economic reasons, these are preferably used in an impure state as waste products from other processes, e.g. in the form of milk product media, molasses or sulphite waste sludge. Fats can also be used for this purpose, but carbohydrates are preferred. As is the case with the source of nitrogen, the final choice here too will depend on the balance of benefits under the circumstances.

The nutritious salts that should be present include phosphates in particular. It is also preferred to have a source for chloride ions and magnesium ions present, especially if the other nutrients used have too little content of available chlorine and magnesium.

It has been found that the fundamentally most important factor for a successful execution of the method is regulation of the amount of available nitrogen in the medium. It has been found that it is possible to prepare griseofulvin from media whose content of available nitrogen varies within a large area, but it has also been found that this content prominently affects the yield of griseofulvin and that in order to achieve industrially satisfactory yields it must be carefully regulated and must almost be kept lower than one might expect.

The invention therefore relates to a method for the production of griseofulvin by cultivating a griseofulvin-producing organism in connection with a culture medium which supports the growth of this organism during the formation of griseofulvin and which contains assimilable sources of nitrogen, sources of carbon and energy, as well as nutritious salts , and the invention is characterized in that the cultivation is carried out under submerged, aerobic conditions and in that the available nitrogen in the assimilable source for nitrogen is set to a ratio of 0.04 to 0.30%.

It has been found that to achieve optimum results the content of available nitrogen should preferably be between about 0.075% and 0.25%. However, it has also been found that the optimum nitrogen content varies somewhat with the type of inoculation medium and the type of culture apparatus used. Thus, e.g. in 250 ml shaking flasks the nitrogen content of the culture medium is optimally around 0.2% when a spore suspension is used as inoculation medium, but optimally around 0.1-0.15% when a 5% vegetable inoculation medium is used. Very satisfactory yields of griseofulvin have been achieved in 680 liter cultivation devices with a nitrogen content of 0.2% in the cultivation medium and a vegetable inoculation medium. These variations in the optimum nitrogen content can be seen from the following table. In the above and following tables, "Ak" means activity found during examination.

The media used in carrying out the cultivations of which the results are indicated in the above table were the following:

Solids from grain liquor 0.1%, 0.17% or 0.24% N. Lactose, glucose, sucrose or starch 7%.

The following factors are also important for obtaining optimal results when carrying out the method according to the invention: (a) As mentioned above, it is preferred

to use a carbohydrate as a source of carbon and energy. The carbohydrate content should preferably be higher than one might expect. The carbohydrate content can be as low as 3.5% or even lower, but it is preferred to keep it above 5%. The upper limit is not critical, but normally it does not exceed around 12%. When the coal hydrate is used in impure form, e.g. like molasses, these percentage values refer to the carbohydrate content of the final medium. Correspondingly larger quantities of the impure carbohydrate-containing material must therefore be added. As mentioned above, all carbohydrate sources that are commonly used in cultivation processes can be used in the present method. Among these are glucose, brown sugar and starch, but currently it is preferred to use lac-

crazy. However, it has been found that the beneficial effect of lactose only shows itself when a vegetative inoculation medium is used, which can also be seen from the above Table A.

The beneficial results achieved with high carbohydrate content can be seen quantitatively from the following table B:

The results given in this table were obtained by using a vegetative inoculation medium in the following medium: C.S.L. solids corresponding to 0.17% N. Lactose, glucose, sucrose and starch 7% or 3.5%

(b) The medium's pH value is not critical but should preferably be set to between 4.5 and 5.5 before cultivation begins. The pH value rises during the course of cultivation and when it is complete, the pH value is

often around 6.5 to 8. Griseofulvin is stable within the pH range of 3.0 to 8.8. In order to achieve the necessary initial setting of the pH value and to have a buffer effect during cultivation, it has been found advantageous to add to the cultivation medium between 0.4% and 1.2% phosphate and limestone or lime. It is currently preferred to use 0.4 to 0.8% potassium dihydrogen phosphate and 0.8% limestone. It is noted that these substances also act as nutritious salts and that the phosphate in particular has a very beneficial effect on the yield of griseofulvin. The presence of calsten significantly reduces pigmentation in the culture.

The following two tables C and D illustrate the effects of phosphate and limestone.

These results were obtained using the lactose C.S.K medium I described below and from the results it will be seen that the titers react very favorably to the presence of phosphate, but only quite a bit to limestone when the carbohydrate source is lactose. However, an undesirable formation of brown pigment occurred when limestone was not present. The following table shows that the good effects of the limestone on the titer are much more prominent when using a culture medium similar to the above with the exception that the lactose is replaced by glucose and that it contains two different sources of phosphate in a quantity ratio of 0, 4% in each case. Here too, reduce the presence of limestone pigment formation. (c) The degree of aeration should be high. It generally turns out that the stronger the aeration, the higher the yield of griseofulvin will be, within certain limits, and this is particularly the case when the nitrogen content in the medium is high. However, it is not possible to specify exact values for the aeration since, as is known, the degree of aeration is not only dependent on the ratio in which the air is supplied, but also on the efficiency with which it is used, which in turn depends on many other variables which are too numerous so that they can be enumerated. Among these are the size and shape of the cultivation vessels and the way in which the air is introduced. The effect of increasing the aeration is shown in the following table E. In this table are given the results of a series of cultivations carried out using media in which the nitrogen content was in each case 0.15%, while the carbohydrate source varied in the different media. Portions of 40 ml, 60 ml and 80 ml of culture medium were in each case shaken in 250 ml flasks. It will be understood that the smaller the volume of the culture, the greater the aeration. When excluding the carbohydrate source, the medium used in each case was similar to C.S.L.-lactose medium I, which is described below. The media were inoculated with 1 ml, 1.5 ml and 2 ml of a submerged spore suspension, respectively.

(d) The medium must contain available

chlorine to avoid the formation of dechlor-griseofulvin. Provided that there is sufficient chlorine present, the quantity ratio of chlorine does not seem to be critical. Complex organic materials that can be used as a nitrogen source generally contain some chlorine. However, it has been found that to ensure the achievement of satisfactory results it is generally appropriate to add between 0.05 and 0.25% of a suitable soluble chloride. Currently, it is preferred to use about 0.1% KC1.

(e) The inoculation medium used may

be a spore suspension or a free-wax

end vegetative inoculation medium. It is preferable to use the latter. When a spore suspension is used for inoculation in a 150 liter vessel for the preparation of inoculation medium, it has been found satisfactory to use 150 ml of suspension with a spore count of about 18 x 10" spores per ml. When this vegetative inoculation medium is well developed, it is used in a proportion between 1 and 10% for inoculation of the culture medium.

In the following, three media are given as examples which have proven to be satisfactory for the production step of the method.

(a) Medium for cultivation under submerged conditions. (I)

b) Medium for cultivation under submerged conditions (II) c) Medium for cultivation under submerged conditions (III)

The temperature at which the cultivation is carried out is not remarkably critical. Currently, it is preferred to use a temperature of around 25° C. It is often advantageous to add an anti-foaming agent during cultivation, such as, for example f heritageless mineral oil, suitably in a quantity ratio of from 0.25 to 0.75% calculated on the medium.

It has been found that in order to produce spore formation in the griseofulvin-producing mold for the production of spore suspensions suitable for use as an inoculum medium, either for the development stage or for the production stage, it is generally not satisfactory to use the normal production medium . This is particularly the case when using the preferred strain Penicillium patulum Bainier-Thom. However, it has been found that spore suspensions can be formed under submerged conditions provided that certain requirements are met. In particular, it is therefore desirable that the content of total available nitrogen in media such as

is used for the production of spore suspensions, is below 0.25% and preferably

between 0.05 and 0.1% and likewise that good aeration is maintained The latter condition is perhaps the most important. The optimal values for these conditions vary

slightly from one strain to another of the organism.

It has also been found that there is a prominent interaction between the nitrogen content and the aeration. The weaker the aeration, the lower the nitrogen content required. To illustrate this, it is mentioned that it has been found that 600 ml of a medium in which whey is used as a hydrogen source and gives a nitrogen content of about 0.05% and which contains suitable amounts of lactose, phosphate, chloride and grain extract, gave abundant spore formation when it was shaken vigorously in a 2 liter flask at 25° C for 6 days.

For illustrative purposes, an example of a medium which was found to be suitable for the spore formation step of the method is given in the following.

Medium for submerged spore development of P. patulum Bainier Thorn. (IV)

As mentioned above, the spore suspension obtained from the spore formation step can either be used for direct inoculation in the cultivation step itself or it can be subjected to development in any suitable medium so as to provide a vegetative inoculation medium for the cultivation step. The latter route is currently preferred, especially when the carbohydrate source in the cultivation step is lactose. When glucose is used in the cultivation step, the advantage of a vegetative inoculation medium is not so prominent. The conditions that determine the development stage of the method are generally not different from the conditions in the final cultivation stage. It has thus been found that satisfactory results can be obtained with different development media, among other media I and II which are described above for use in the cultivation step of the method.

It has been found that griseofulvin formed

during the cultivation for the majority of the people concerned, e.g. 90%, remains in the mycelium itself instead of passing into the culture medium unless the culture is allowed to go too far. In the latter case, the mycelium breaks down and the griseofulvin can be distributed in the culture medium. Extraction of griseofulvin from the culture medium itself presents difficulties. It is therefore preferred to take advantage of the fact that the griseofulvin is retained in the mycelium and consequently to interrupt cultivation before degradation of the mycelium occurs to any significant extent.

The recovery of the griseofulvin can be carried out in any suitable manner. Methods for carrying out such extractions have previously been proposed in connection with surface cultures and, in general, such methods are also suitable for use in the method according to the present invention.

In the following, an embodiment of a preferred extraction method is described as an example. The mycelium is separated from the cultivation culture, preferably by filtration at a pH value which should be lower than 8.0. The filtrate contains around 10% of the griseofulvin formed and is generally thrown away, but if desired, the antibiotic agent can be recovered from this by a special extraction technique. The solids in the whey are extracted with an organic solvent, e.g. ethyl acetate, no-malt butanol, amyl acetate or preferably butyl acetate. The extraction is conveniently carried out by mixing the mycelium and the solvent, preferably in equal parts by weight and volume respectively, over a period of about 15 minutes. and then separate the residue from the solution by sedimentation and decantation. This extraction operation should preferably be repeated two or more times. The decanted solution is then clarified preferably by centrifugation or filtration, after which it is concentrated, preferably in a vacuum under such conditions that the temperature does not exceed 50° C. The concentration is carried out as far as is practically possible, after which the concentrate is allowed to cool to 20 ° C or thereabouts and the solid material is filtered off. If, as is generally the case, some oily liquid has been extracted from the mycelium together with the griseofulvin, oil adhering to the solid is washed away by mixing this with a suitable solvent such as butyl acetate in equal proportions by weight acc. volume, after which you filter again. The washed solid is preferably dried in a vacuum, e.g. over phosphorus pentoxide at room temperature. The crude griseofulvin thus obtained can be purified by dissolving it in an organic solvent such as acetone, filtering the solution and precipitating the griseofulvin by adding water in which griseofulvin is not soluble to any greater extent. If necessary, these operations can be repeated, after which the product is dried. You then get pure, crystalline griseofulvin.

In the following, an example of a method for producing griseofulvin on a small scale is given for illustrative purposes.

Production of spore suspension. 600 ml of the following medium was autoclaved in a 2 liter conical flask:

The flask was inoculated with a suspension of spores from a Czapek-Dox agar culture of Penicillium patulum Bainier Thom (4640, 455) C.M.I. 39, 809), in which the spores were well developed and incubated on a shaker at 25° C. for 7 days. After this time, the culture had produced copious amounts of down-cultivated spores.

Vegetative cultivation stage. 150 liters of the following medium were inoculated in a culture apparatus and while stirring with 150 ml of the suspension of the submerged spores, containing about 18 x 10" spores per ml.

No regulation of the pH value. Sterilized for 20 min. at 120°C.

Aeration was carried out at a rate of around 85 liters of air per hour. my. in the first 24 hours, after which the aeration was gradually increased to 226 liters of air per my. as the culture progressed and the foaming subsided. The temperature was 25° C and stirring was carried out at a speed of 350 revolutions per minute. my. After 28 hours, this material was transferred to culture medium.

Cultivation stage. 450 liters were used

No regulation of the pH value.

Sterilized 20 min. at 120° C. The culture medium was inoculated with 10%

vegetative inoculation medium from the previous stage.

as close as possible to 283 liters per my. in those

first 8 hours, after which it was increased to around 566 liters per my. The temperature was

25° C and was stirred at a speed

at 350 revolutions per my.

As an anti-foaming agent was used

f heirless mineral oil as required.

The details of the cultivation are as follows:

During the extraction with a solvent using the method described above, 150 g were obtained

griseofulvin with 95% purity.

Claims (6)

1. Method for the production of griseofulvin with the cultivation of a griseofulvin-producing organism in connection with a culture medium which supports the growth of this organism during the formation of griseofulvin and which contains assimilable sources of nitrogen, sources of carbon and energy as well as nutritious salts, characterized in that the cultivation is carried out under submerged aerobic conditions and in that the available nitrogen in the assimilable nitrogen source is set to a ratio of from 0.04 to 0.30%, preferably from 0.075 to 0.25%. 2. Method according to claim 1 where a carbohydrate is used as a source of carbon and energy, characterized in that the carbon hydrate is present in the medium in a quantity ratio of not less than 3.5% and preferably not less than 5.0%. 3. Method according to any one of the preceding claims, characterized in that penicillium patulum Bainier Thorn is used as the griseofulvin-producing organism. 4. Method according to any of the preceding claims where among the nutrient salts in the culture medium phosphates are present, preferably KH2PO+ and K2HPO4, characterized in that the phosphates are present in the medium in a quantity ratio between 0.4 and 0.8%. 5. Method according to any of the preceding claims, where chlorides, preferably potassium chloride, are present among the nutrient salts in the culture medium, characterized in that the chlorides are present in a quantity ratio of at least 0.1%. 6. Method according to any of the preceding claims, characterized in that the griseofulvin-producing organism is grown to the point where the mycelium is broken down before the mycelium is separated from the culture liquid and the griseofulvin is extracted from the mycelium in a known manner by extraction with solvents, preferably butyl acetate.